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1.
Proc Natl Acad Sci U S A ; 111(36): 13034-9, 2014 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-25157169

RESUMO

Microbial rhodopsins are a diverse group of photoactive transmembrane proteins found in all three domains of life. A member of this protein family, Archaerhodopsin-3 (Arch) of halobacterium Halorubrum sodomense, was recently shown to function as a fluorescent indicator of membrane potential when expressed in mammalian neurons. Arch fluorescence, however, is very dim and is not optimal for applications in live-cell imaging. We used directed evolution to identify mutations that dramatically improve the absolute brightness of Arch, as confirmed biochemically and with live-cell imaging (in Escherichia coli and human embryonic kidney 293 cells). In some fluorescent Arch variants, the pK(a) of the protonated Schiff-base linkage to retinal is near neutral pH, a useful feature for voltage-sensing applications. These bright Arch variants enable labeling of biological membranes in the far-red/infrared and exhibit the furthest red-shifted fluorescence emission thus far reported for a fluorescent protein (maximal excitation/emission at ∼ 620 nm/730 nm).


Assuntos
Proteínas Arqueais/metabolismo , Evolução Molecular Direcionada , Sítios de Ligação , Sobrevivência Celular , Escherichia coli/metabolismo , Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Halorubrum/metabolismo , Humanos , Proteínas Mutantes/metabolismo , Mutação , Homologia Estrutural de Proteína
2.
Biochemistry ; 53(48): 7549-61, 2014 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-25375769

RESUMO

A group of microbial retinal proteins most closely related to the proton pump xanthorhodopsin has a novel sequence motif and a novel function. Instead of, or in addition to, proton transport, they perform light-driven sodium ion transport, as reported for one representative of this group (KR2) from Krokinobacter. In this paper, we examine a similar protein, GLR from Gillisia limnaea, expressed in Escherichia coli, which shares some properties with KR2 but transports only Na(+). The absorption spectrum of GLR is insensitive to Na(+) at concentrations of ≤3 M. However, very low concentrations of Na(+) cause profound differences in the decay and rise time of photocycle intermediates, consistent with a switch from a "Na(+)-independent" to a "Na(+)-dependent" photocycle (or photocycle branch) at ∼60 µM Na(+). The rates of photocycle steps in the latter, but not the former, are linearly dependent on Na(+) concentration. This suggests that a high-affinity Na(+) binding site is created transiently after photoexcitation, and entry of Na(+) from the bulk to this site redirects the course of events in the remainder of the cycle. A greater concentration of Na(+) is needed for switching the reaction path at lower pH. The data suggest therefore competition between H(+) and Na(+) to determine the two alternative pathways. The idea that a Na(+) binding site can be created at the Schiff base counterion is supported by the finding that upon perturbation of this region in the D251E mutant, Na(+) binds without photoexcitation. Binding of Na(+) to the mutant shifts the chromophore maximum to the red like that of H(+), which occurs in the photocycle of the wild type.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/efeitos da radiação , Flavobacteriaceae/enzimologia , ATPase Trocadora de Sódio-Potássio/metabolismo , ATPase Trocadora de Sódio-Potássio/efeitos da radiação , Sequência de Aminoácidos , Substituição de Aminoácidos , Ácido Aspártico/química , Proteínas de Bactérias/genética , Sítios de Ligação , Flavobacteriaceae/genética , Flavobacteriaceae/efeitos da radiação , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Processos Fotoquímicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/efeitos da radiação , Rodopsinas Microbianas/genética , Rodopsinas Microbianas/metabolismo , Rodopsinas Microbianas/efeitos da radiação , Bases de Schiff/química , Homologia de Sequência de Aminoácidos , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/genética , Espectroscopia de Infravermelho com Transformada de Fourier
3.
J Biol Chem ; 288(29): 21254-21265, 2013 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-23696649

RESUMO

A lysine instead of the usual carboxyl group is in place of the internal proton donor to the retinal Schiff base in the light-driven proton pump of Exiguobacterium sibiricum (ESR). The involvement of this lysine in proton transfer is indicated by the finding that its substitution with alanine or other residues slows reprotonation of the Schiff base (decay of the M intermediate) by more than 2 orders of magnitude. In these mutants, the rate constant of the M decay linearly decreases with a decrease in proton concentration, as expected if reprotonation is limited by the uptake of a proton from the bulk. In wild type ESR, M decay is biphasic, and the rate constants are nearly pH-independent between pH 6 and 9. Proton uptake occurs after M formation but before M decay, which is especially evident in D2O and at high pH. Proton uptake is biphasic; the amplitude of the fast phase decreases with a pKa of 8.5 ± 0.3, which reflects the pKa of the donor during proton uptake. Similarly, the fraction of the faster component of M decay decreases and the slower one increases, with a pKa of 8.1 ± 0.2. The data therefore suggest that the reprotonation of the Schiff base in ESR is preceded by transient protonation of an initially unprotonated donor, which is probably the ε-amino group of Lys-96 or a water molecule in its vicinity, and it facilitates proton delivery from the bulk to the reaction center of the protein.


Assuntos
Proteínas de Bactérias/metabolismo , Halobacterium/metabolismo , Luz , Lisina/metabolismo , Prótons , Bases de Schiff/metabolismo , Absorção/efeitos da radiação , Alanina/genética , Substituição de Aminoácidos/genética , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/efeitos da radiação , Óxido de Deutério/metabolismo , Escherichia coli/metabolismo , Halobacterium/efeitos dos fármacos , Halobacterium/efeitos da radiação , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Concentração de Íons de Hidrogênio/efeitos da radiação , Cinética , Lipossomos/metabolismo , Lisina/genética , Proteínas Mutantes/metabolismo , Azida Sódica/farmacologia , Fatores de Tempo
4.
J Photochem Photobiol B ; 234: 112529, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35878544

RESUMO

Light-driven proton transport by microbial retinal proteins such as archaeal bacteriorhodopsin involves carboxylic residues as internal proton donors to the catalytic center which is a retinal Schiff base (SB). The proton donor, Asp96 in bacteriorhodopsin, supplies a proton to the transiently deprotonated Schiff base during the photochemical cycle. Subsequent proton uptake resets the protonated state of the donor. This two step process became a distinctive signature of retinal based proton pumps. Similar steps are observed also in many natural variants of bacterial proteorhodopsins and xanthorhodopsins where glutamic acid residues serve as a proton donor. Recently, however, an exception to this rule was found. A retinal protein from Exiguobacterium sibiricum, ESR, contains a Lys residue in place of Asp or Glu, which facilitates proton transfer from the bulk to the SB. Lys96 can be functionally replaced with the more common donor residues, Asp or Glu. Proton transfer to the SB in the mutants containing these replacements (K96E and K96D/A47T) is much faster than in the proteins lacking the proton donor (K96A and similar mutants), and in the case of K96D/A47T, comparable with that in the wild type, indicating that carboxylic residues can replace Lys96 as proton donors in ESR. We show here that there are important differences in the functioning of these residues in ESR from the way Asp96 functions in bacteriorhodopsin. Reprotonation of the SB and proton uptake from the bulk occur almost simultaneously during the M to N transition (as in the wild type ESR at neutral pH), whereas in bacteriorhodopsin these two steps are well separated in time and occur during the M to N and N to O transitions, respectively.


Assuntos
Bacteriorodopsinas , Prótons , Bacteriorodopsinas/química , Exiguobacterium , Concentração de Íons de Hidrogênio , Bombas de Próton/química , Bombas de Próton/metabolismo , Bases de Schiff/química
5.
J Membr Biol ; 239(1-2): 95-104, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21104180

RESUMO

Salinixanthin, a C(40)-carotenoid acyl glycoside, serves as a light-harvesting antenna in the retinal-based proton pump xanthorhodopsin of Salinibacter ruber. In the crystallographic structure of this protein, the conjugated chain of salinixanthin is located at the protein-lipid boundary and interacts with residues of helices E and F. Its ring, with a 4-keto group, is rotated relative to the plane of the π-system of the carotenoid polyene chain and immobilized in a binding site near the ß-ionone retinal ring. We show here that the carotenoid can be removed by oxidation with ammonium persulfate, with little effect on the other chromophore, retinal. The characteristic CD bands attributed to bound salinixanthin are now absent. The kinetics of the photocycle is only slightly perturbed, showing a 1.5-fold decrease in the overall turnover rate. The carotenoid-free protein can be reconstituted with salinixanthin extracted from the cell membrane of S. ruber. Reconstitution is accompanied by restoration of the characteristic vibronic structure of the absorption spectrum of the antenna carotenoid, its chirality, and the excited-state energy transfer to the retinal. Minor modification of salinixanthin, by reducing the carbonyl C=O double bond in the ring to a C-OH, suppresses its binding to the protein and eliminates the antenna function. This indicates that the presence of the 4-keto group is critical for carotenoid binding and efficient energy transfer.


Assuntos
Proteínas de Bactérias/química , Carotenoides/química , Rodopsinas Microbianas/química , Sulfato de Amônio/química , Proteínas de Bactérias/metabolismo , Carotenoides/metabolismo , Transferência de Energia , Glicosídeos/metabolismo , Cinética , Oxirredução , Rodopsinas Microbianas/metabolismo
6.
Proc Natl Acad Sci U S A ; 105(43): 16561-5, 2008 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-18922772

RESUMO

Homologous to bacteriorhodopsin and even more to proteorhodopsin, xanthorhodopsin is a light-driven proton pump that, in addition to retinal, contains a noncovalently bound carotenoid with a function of a light-harvesting antenna. We determined the structure of this eubacterial membrane protein-carotenoid complex by X-ray diffraction, to 1.9-A resolution. Although it contains 7 transmembrane helices like bacteriorhodopsin and archaerhodopsin, the structure of xanthorhodopsin is considerably different from the 2 archaeal proteins. The crystallographic model for this rhodopsin introduces structural motifs for proton transfer during the reaction cycle, particularly for proton release, that are dramatically different from those in other retinal-based transmembrane pumps. Further, it contains a histidine-aspartate complex for regulating the pK(a) of the primary proton acceptor not present in archaeal pumps but apparently conserved in eubacterial pumps. In addition to aiding elucidation of a more general proton transfer mechanism for light-driven energy transducers, the structure defines also the geometry of the carotenoid and the retinal. The close approach of the 2 polyenes at their ring ends explains why the efficiency of the excited-state energy transfer is as high as approximately 45%, and the 46 degrees angle between them suggests that the chromophore location is a compromise between optimal capture of light of all polarization angles and excited-state energy transfer.


Assuntos
Proteínas de Bactérias/química , Transferência de Energia , Eubacterium/química , Luz , Rodopsinas Microbianas/química , Carotenoides/química , Cristalografia por Raios X , Conformação Proteica , Prótons , Retinaldeído/química
7.
Biochemistry ; 49(45): 9792-9, 2010 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-20942439

RESUMO

In previous work, we reconstituted salinixanthin, the C(40)-carotenoid acyl glycoside that serves as a light-harvesting antenna to the light-driven proton pump xanthorhodopsin, into a different protein, gloeobacter rhodopsin expressed in Escherichia coli, and demonstrated that it transfers energy to the retinal chromophore [Imasheva, E. S., et al. (2009) Biochemistry 48, 10948]. The key to binding of salinixanthin was the accommodation of its ring near the retinal ß-ionone ring. Here we examine two questions. Do any of the native Gloeobacter carotenoids bind to gloeobacter rhodopsin, and does the 4-keto group of the ring play a role in binding? There is no salinixanthin in Gloeobacter violaceous, but a simpler carotenoid, echinenone, also with a 4-keto group but lacking the acyl glycoside, is present in addition to ß-carotene and oscillol. We show that ß-carotene does not bind to gloeobacter rhodopsin, but its 4-keto derivative, echinenone, does and functions as a light-harvesting antenna. This indicates that the 4-keto group is critical for carotenoid binding. Further evidence of this is the fact that salinixanthol, an analogue of salinixanthin in which the 4-keto group is reduced to hydroxyl, does not bind and is not engaged in energy transfer. According to the crystal structure of xanthorhodopsin, the ring of salinixanthin in the binding site is turned out of the plane of the polyene conjugated chain. A similar conformation is expected for echinenone in the gloeobacter rhodopsin. We suggest that the 4-keto group in salinixanthin and echinenone allows for the twisted conformation of the ring around the C6-C7 bond and probably is engaged in an interaction that locks the carotenoid in the binding site.


Assuntos
Carotenoides/química , Rodopsina/química , Rodopsinas Microbianas/química , Xantina/química , Carotenoides/metabolismo , Dicroísmo Circular , Escherichia coli/genética , Escherichia coli/metabolismo , Glicosídeos/química , Modelos Moleculares , Rodopsina/genética , Rodopsinas Microbianas/metabolismo , Espectrofotometria , beta Caroteno/química
8.
Biophys J ; 96(6): 2268-77, 2009 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-19289053

RESUMO

Xanthorhodopsin of the extremely halophilic bacterium Salinibacter ruber represents a novel antenna system. It consists of a carbonyl carotenoid, salinixanthin, bound to a retinal protein that serves as a light-driven transmembrane proton pump similar to bacteriorhodopsin of archaea. Here we apply the femtosecond transient absorption technique to reveal the excited-state dynamics of salinixanthin both in solution and in xanthorhodopsin. The results not only disclose extremely fast energy transfer rates and pathways, they also reveal effects of the binding site on the excited-state properties of the carotenoid. We compared the excited-state dynamics of salinixanthin in xanthorhodopsin and in NaBH(4)-treated xanthorhodopsin. The NaBH(4) treatment prevents energy transfer without perturbing the carotenoid binding site, and allows observation of changes in salinixanthin excited-state dynamics related to specific binding. The S(1) lifetimes of salinixanthin in untreated and NaBH(4)-treated xanthorhodopsin were identical (3 ps), confirming the absence of the S(1)-mediated energy transfer. The kinetics of salinixanthin S(2) decay probed in the near-infrared region demonstrated a change of the S(2) lifetime from 66 fs in untreated xanthorhodopsin to 110 fs in the NaBH(4)-treated protein. This corresponds to a salinixanthin-retinal energy transfer time of 165 fs and an efficiency of 40%. In addition, binding of salinixanthin to xanthorhodopsin increases the population of the S(*) state that decays in 6 ps predominantly to the ground state, but a small fraction (<10%) of the S(*) state generates a triplet state.


Assuntos
Proteínas de Bactérias/química , Carotenoides/química , Transferência de Energia , Glicosídeos/química , Rodopsinas Microbianas/química , Absorção , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Boroidretos/farmacologia , Carotenoides/metabolismo , Glicosídeos/metabolismo , Cinética , Metanol , Rodopsinas Microbianas/metabolismo , Análise Espectral
9.
Biochemistry ; 48(46): 10948-55, 2009 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-19842712

RESUMO

We show that salinixanthin, the light-harvesting carotenoid antenna of xanthorhodopsin, can be reconstituted into the retinal protein from Gloeobacter violaceus expressed in Escherichia coli. Reconstitution of gloeobacter rhodopsin with the carotenoid is accompanied by characteristic absorption changes and the appearance of CD bands similar to those observed for xanthorhodopsin that indicate immobilization and twist of the carotenoid in the binding site. As in xanthorhodopsin, the carotenoid functions as a light-harvesting antenna. The excitation spectrum for retinal fluorescence emission shows that ca. 36% of the energy absorbed by the carotenoid is transferred to the retinal. From excitation anisotropy, we calculate the angle between the two chromophores as being ca. 50 degrees , similar to that in xanthorhodopsin. The results indicate that gloeobacter rhodopsin binds salinixanthin in a manner similar to that of xanthorhodopsin and suggest that it might bind a carotenoid also in vivo. In the crystallographic structure of xanthorhodopsin, the conjugated chain of the carotenoid lies on the surface of helices E and F, and the 4-keto ring is immersed in the protein at van der Waals distance from the ionone ring of the retinal. The 4-keto ring is in the space occupied by a tryptophan in bacteriorhodopsin, which is replaced by the smaller glycine in xanthorhodopsin and gloeobacter rhodopsin. Specific binding of the carotenoid and its light-harvesting function are eliminated by a single mutation of the gloeobacter protein that replaces this glycine with a tryptophan. This indicates that the 4-keto ring is critically involved in carotenoid binding and suggests that a number of other recently identified retinal proteins, from a diverse group of organisms, could also contain carotenoid antenna since they carry the homologous glycine near the retinal.


Assuntos
Carotenoides/metabolismo , Glicosídeos/metabolismo , Rodopsinas Microbianas/química , Rodopsinas Microbianas/metabolismo , Substituição de Aminoácidos/genética , Bacteroidetes/química , Sítios de Ligação/genética , Carotenoides/química , Dicroísmo Circular , Cianobactérias/genética , Polarização de Fluorescência , Glicosídeos/química , Hidroxilamina/química , Conformação Molecular , Ligação Proteica/fisiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Retinaldeído/química , Rodopsinas Microbianas/genética , Bases de Schiff/química , Espectrometria de Fluorescência , Espectrofotometria
10.
Biophys J ; 95(5): 2402-14, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18515390

RESUMO

The cell membrane of Salinibacter ruber contains xanthorhodopsin, a light-driven transmembrane proton pump with two chromophores: a retinal and the carotenoid, salinixanthin. Action spectra for transport had indicated that light absorbed by either is utilized for function. If the carotenoid is an antenna in this protein, its excited state energy has to be transferred to the retinal and should be detected in the retinal fluorescence. From fluorescence studies, we show that energy transfer occurs from the excited singlet S(2) state of salinixanthin to the S(1) state of the retinal. Comparison of the absorption spectrum with the excitation spectrum for retinal emission yields 45 +/- 5% efficiency for the energy transfer. Such high efficiency would require close proximity and favorable geometry for the two polyene chains, but from the heptahelical crystallographic structure of the homologous retinal protein, bacteriorhodopsin, it is not clear where the carotenoid can be located near the retinal. The fluorescence excitation anisotropy spectrum reveals that the angle between their transition dipole moments is 56 +/- 3 degrees . The protein accommodates the carotenoid as a second chromophore in a distinct binding site to harvest light with both extended wavelength and polarization ranges. The results establish xanthorhodopsin as the simplest biological excited-state donor-acceptor system for collecting light.


Assuntos
Proteínas de Bactérias/química , Bacteroidetes/química , Carotenoides/química , Transferência de Energia , Glicosídeos/química , Retinaldeído/química , Rodopsina/química , Rodopsinas Microbianas/química , Polarização de Fluorescência , Conformação Proteica , Análise Espectral Raman
11.
Photochem Photobiol ; 84(4): 977-84, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18399915

RESUMO

Xanthorhodopsin is a light-driven proton pump in the extremely halophilic bacterium Salinibacter ruber. Its unique feature is that besides retinal it has a carotenoid, salinixanthin, with a light harvesting function. Tight and specific binding of the carotenoid antenna is controlled by binding of the retinal. Addition of all-trans retinal to xanthorhodopsin bleached with hydroxylamine restores not only the retinal chromophore absorption band, but causes sharpening of the salinixanthin bands reflecting its rigid binding by the protein. In this report we examine the correlation of the changes in the two chromophores during bleaching and reconstitution with native all-trans retinal, artificial retinal analogs and retinol. Bleaching and reconstitution both appear to be multistage processes. The carotenoid absorption changes during bleaching occurred not only upon hydrolysis of the Schiff base but continued while the retinal was leaving its binding site. In the case of reconstitution, the 13-desmethyl analog formed the protonated Schiff base slower than retinal, and provided the opportunity to observe changes in carotenoid binding at various stages. The characteristic sharpening of the carotenoid bands, indicative of its reduced conformational heterogeneity in the binding site, occurs when the retinal occupies the binding site but the covalent bond to Lys-240 via a Schiff base is not yet formed. This is confirmed by the results for retinol reconstitution, where the Schiff base does not form but the carotenoid exhibits its characteristic spectral change from the binding.


Assuntos
Proteínas de Bactérias/química , Eubacterium/química , Retinaldeído/química , Rodopsina/química , Cinética , Rodopsinas Microbianas , Bases de Schiff , Espectrofotometria
12.
Photochem Photobiol ; 84(4): 880-8, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18346087

RESUMO

Pharaonis phoborhodopsin (ppR), a negative phototaxis receptor of Natronomonas pharaonis, undergoes photocycle similar to the light-driven proton pump bacteriorhodopsin (BR), but the turnover rate is much slower due to much longer lifetimes of the M and O intermediates. The M decay was shown to become as fast as it is in BR in the L40T/F86D mutant. We examined the effects of hydrostatic pressure on the decay of these intermediates. For BR, pressure decelerated M decay but slightly affected O decay. In contrast, with ppR and with its L40T/F86D mutant, pressure slightly affected M decay but accelerated O decay. Clearly, the pressure-dependent factors for M and O decay are different in BR and ppR. In order to examine the deprotonation of Asp75 in unphotolyzed ppR we performed stopped flow experiments. The pH jump-induced deprotonation of Asp75 occurred with 60 ms, which is at least 20 times slower than deprotonation of the equivalent Asp85 in BR and about 10-fold faster than the O decay of ppR. These data suggest that proton transfer is slowed not only in the cytoplasmic channel but also in the extracellular channel of ppR and that the light-induced structural changes in the O intermediate of ppR additionally decrease this rate.


Assuntos
Halorrodopsinas/química , Natronobacterium/química , Rodopsinas Sensoriais/química , Ácido Aspártico/análise , Bacteriorodopsinas/química , Bacteriorodopsinas/efeitos da radiação , Halorrodopsinas/efeitos da radiação , Pressão Hidrostática , Cinética , Luz , Natronobacterium/efeitos da radiação , Fotólise , Prótons , Rodopsinas Sensoriais/efeitos da radiação , Espectrofotometria
13.
Photochem Photobiol ; 82(6): 1406-13, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16649816

RESUMO

Xanthorhodopsin (XR), the light-driven proton pump of the halophilic eubacterium Salinibacter ruber, exhibits substantial homology to bacteriorhodopsin (BR) of archaea and proteorhodopsin (PR) of marine bacteria, but unlike them contains a light-harvesting carotenoid antenna, salinixanthin, as well as retinal. We report here the pH-dependent properties of XR. The pKa of the retinal Schiff base is as high as in BR, i.e. > or =12.4. Deprotonation of the Schiff base and the ensuing alkaline denaturation cause large changes in the absorption bands of the carotenoid antenna, which lose intensity and become broader, making the spectrum similar to that of salinixanthin not bound to XR. A small redshift of the retinal chromophore band and increase of its extinction, as well as the pH-dependent amplitude of the M intermediate indicate that in detergent-solubilized XR the pKa of the Schiff base counterion and proton acceptor is about 6 (compared to 2.6 in BR, and 7.5 in PR). The protonation of the counterion is accompanied by a small blueshift of the carotenoid absorption bands. The pigment is stable in the dark upon acidification to pH 2. At pH < 2 a transition to a blueshifted species absorbing around 440 nm occurs, accompanied by loss of resolution of the carotenoid absorption bands. At pH < 3 illumination of XR with continuous light causes accumulation of long-lived photoproduct(s) with an absorption maximum around 400 nm. The photocycle of XR was examined between pH 4 and 10 in solubilized samples. The pH dependence of recovery of the initial state slows at both acid and alkaline pH, with pKas of 6.0 and 9.3. The decrease in the rates with pKa 6.0 is apparently caused by protonation of the counterion and proton acceptor, and that at high pH reflects the pKa of the internal proton donor, Glu94, at the times in the photocycle when this group equilibrates with the bulk.


Assuntos
Proteínas de Bactérias/química , Concentração de Íons de Hidrogênio , Rodopsina/química , Carotenoides/química , Eubacterium/química , Rodopsinas Microbianas , Bases de Schiff , Espectrofotometria
14.
Biochemistry ; 45(36): 10998-1004, 2006 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-16953586

RESUMO

In xanthorhodopsin, a retinal protein-carotenoid complex of Salinibacter ruber, the carotenoid salinixanthin functions as a light-harvesting antenna in supplying additional excitation energy for retinal isomerization and proton transport. Another retinal protein, archaerhodopsin, has been shown to contain a carotenoid, bacterioruberin, but without an antenna function. We report here that the binding site confers a chiral geometry on salinixanthin in xanthorhodopsin and confirm that the same is true for bacterioruberin in archaerhodopsin. Cell membranes containing these rhodopsins exhibit CD spectra with sharp positive bands in the visible region where the carotenoids absorb, and in the case of xanthorhodopsin a negative band at 536 nm, as well as bands in the UV region. The carotenoid in ethanol has very weak optical activity in the visible region of the spectrum. Denaturation of the opsin upon deprotonation of the Schiff base at pH 12.5 eliminates the induced CD bands in both proteins. In one of these proteins, but not in the other, the carotenoid binding site depends entirely on the retinal. Hydrolysis of the retinal Schiff base of xanthorhodopsin with hydroxylamine eliminates the induced CD bands of salinixanthin. In contrast, hydrolysis of the Schiff base in archaerhodopsin does not abolish the CD bands of bacterioruberin. Thus, consistent with its antenna function, the carotenoid binding site interacts closely with the retinal only in xanthorhodopsin, and this interaction is the major source of the CD bands. In this protein, protonation of the counterion with a decrease in pH from 8 to 5 causes significant changes in the CD spectrum. The observed spectral features suggest that binding of salinixanthin in xanthorhodopsin involves the cyclohexenone ring of the carotenoid and its conformational heterogeneity is restricted.


Assuntos
Carotenoides/química , Glicosídeos/química , Retinaldeído/química , Rodopsinas Microbianas/química , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Bacteroidetes/metabolismo , Carotenoides/metabolismo , Dicroísmo Circular , Glicosídeos/metabolismo , Halobacteriaceae/fisiologia , Concentração de Íons de Hidrogênio , Hidroxilamina/química , Luz , Retinaldeído/metabolismo , Rodopsinas Microbianas/metabolismo , Bases de Schiff
15.
Science ; 309(5743): 2061-4, 2005 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-16179480

RESUMO

Energy transfer from light-harvesting carotenoids to chlorophyll is common in photosynthesis, but such antenna pigments have not been observed in retinal-based ion pumps and photoreceptors. Here we describe xanthorhodopsin, a proton-pumping retinal protein/carotenoid complex in the eubacterium Salinibacter ruber. The wavelength dependence of the rate of pumping and difference absorption spectra measured under a variety of conditions indicate that this protein contains two chromophores, retinal and the carotenoid salinixanthin, in a molar ratio of about 1:1. The two chromophores interact strongly, and light energy absorbed by the carotenoid is transferred to the retinal with a quantum efficiency of approximately 40%. The antenna carotenoid extends the wavelength range of the collection of light for uphill transmembrane proton transport.


Assuntos
Bacteroidetes/química , Complexos de Proteínas Captadores de Luz/química , Bombas de Próton/química , Rodopsinas Microbianas/química , Sequência de Aminoácidos , Bacteroidetes/metabolismo , Carotenoides/química , Carotenoides/metabolismo , Transferência de Energia , Glicosídeos/química , Glicosídeos/metabolismo , Concentração de Íons de Hidrogênio , Hidroxilamina/farmacologia , Luz , Complexos de Proteínas Captadores de Luz/isolamento & purificação , Complexos de Proteínas Captadores de Luz/metabolismo , Espectrometria de Massas , Dados de Sequência Molecular , Consumo de Oxigênio , Bombas de Próton/isolamento & purificação , Bombas de Próton/metabolismo , Retinaldeído/química , Retinaldeído/metabolismo , Rodopsinas Microbianas/isolamento & purificação , Rodopsinas Microbianas/metabolismo , Espectrofotometria Ultravioleta , Análise Espectral
16.
Biochemistry ; 44(32): 10828-38, 2005 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-16086585

RESUMO

Proteorhodopsin, a retinal protein of marine proteobacteria similar to bacteriorhodopsin of the archaea, is a light-driven proton pump. Absorption of a light quantum initiates a reaction cycle (turnover time of ca. 50 ms), which includes photoisomerization of the retinal from the all-trans to the 13-cis form and transient deprotonation of the retinal Schiff base, followed by recovery of the initial state. We report here that in addition to this fast cyclic conversion, illumination at high pH results in accumulation of a long-lived photoproduct absorbing at 362 nm. This photoconversion is much more efficient in the D227N mutant in which the anionic Asp227, which together with Asp97 constitutes the Schiff base counterion, is replaced with a neutral residue. Upon illumination at pH 8.5, most of the D227N pigment is converted to the 362 nm species, with a quantum efficiency of ca. 0.2. The pK(a) for this transition in the wild type is 9.6, but decreased to 7.5 after mutation of Asp227. The short wavelength of the absorption maximum of the photoproduct indicates that it has a deprotonated Schiff base. In the dark, this photoproduct is converted back to the initial pigment with a time constant of 30 min (in D227N, at pH 8.5), but it can be reconverted more rapidly by illumination with near-UV light. Experiments with "locked" retinal analogues which selectively exclude rotation around either the C9=C10, C11=C12, or C13=C14 bond show that formation of the 362 nm species involves isomerization around the C13=C14 bond. In agreement with this, retinal extraction indicates that the 362 nm photoproduct is 13-cis whereas the initial state is predominantly all-trans. A rapid shift of the pH from 8.5 to 4 greatly accelerates thermal reconversion of the 362 nm species to the initial pigment, suggesting that its recovery involving the thermal isomerization of the chromophore is controlled by ionizable residues, primarily the Schiff base and Asp97. The transformation to the long-lived 362 nm photoproduct is apparently a side reaction of the photocycle, a response to high pH, caused by alteration of the normal reprotonation and reisomerization pathway of the Schiff base.


Assuntos
Mutação de Sentido Incorreto , Fotoquímica , Fótons , Rodopsina/química , Bases de Schiff/química , Ácido Aspártico/genética , Meia-Vida , Concentração de Íons de Hidrogênio , Rodopsina/genética , Rodopsinas Microbianas , Raios Ultravioleta
17.
Biochemistry ; 43(6): 1648-55, 2004 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-14769042

RESUMO

Similarly to bacteriorhodopsin, proteorhodopsin that normally contains all-trans and 13-cis retinal is transformed at low pH to a species containing 9-cis retinal under continuous illumination at lambda > 530 nm. This species, absorbing around 430 nm, returns thermally in tens of minutes to initial pigment and can be reconverted also with blue-light illumination. The yield of the 9-cis species is negligibly small at neutral pH but increases manyfold (>100) at acid pH with a pK(a) of 2.6. This indicates that protonation of acidic group(s) alters the photoreaction pathway that leads normally to all-trans --> 13-cis isomerization. In the D97N mutant, in which one of the two acidic groups in the vicinity of the retinal Schiff base is not ionizable, the yield of 9-cis species at low pH shows a pH dependence similar to that in the wild-type but with a somewhat increased pK(a) of 3.3. In contrast to this relatively minor effect, replacement of the other acidic group, Asp227, with Asn results in a remarkable, more than 50-fold, increase in the yield of the light-induced formation of 9-cis species in the pH range 4-6. It appears that protonation of Asp227 at low pH is what causes the dramatic increase in the yield of the 9-cis species in wild-type proteorhodopsin. We conclude that the photoisomerization pathways in proteorhodopsin to 13-cis or 9-cis photoproducts are controlled by the charge state of Asp227.


Assuntos
Ácido Aspártico/química , Luz , Retinaldeído/química , Rodopsina/química , Substituição de Aminoácidos/genética , Asparagina/genética , Ácido Aspártico/genética , Bacteriorodopsinas/química , Bacteriorodopsinas/genética , Bacteriorodopsinas/metabolismo , Sítios de Ligação/genética , Escuridão , Gammaproteobacteria , Concentração de Íons de Hidrogênio , Isomerismo , Rodopsina/genética , Rodopsina/metabolismo , Rodopsinas Microbianas , Solubilidade , Espectroscopia de Infravermelho com Transformada de Fourier , Temperatura
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